The overall goal of this proposal is to test the hypothesis that IERSR can be pharmacologically targeted for cancer therapy. Solid tumors are poorly vascularized and therefore cannot receive sufficient oxygen and nutrients particularly in the least vascularized regions. This causes accumulation of unfolded proteins in endoplasmic reticulum (ER), termed ER-stress. Activation of integrated ER-stress response (IERSR) critically contributes to tumor growth and survival. The IERSR involves inhibiting translation initiation to reduce the demand on the folding capacity of the ER and activating a transcription program to enlarge the size and the folding capacity of the ER. Translation initiation is inhibited through activation of protein kinase R (PKR)-like ER resident kinase PERK and phosphorylation of eukaryotic translation initiation factor 2 aplha (eIF2a). The transcription program to increase the size and the folding capacity of the ER is accomplished by activating key transcription factors such as X box binding protein-1 Xbp-1 that control expression of ER-chaperons, ER biogenesis and ER-associated retrograde protein transport and degradation genes. However, IERSR must be regulated in a spatial and temporal manner because either the failure to activate IERSR or sustained activation of IERSR will reduce survival of stressed cells. We hypothesize that tumor cells utilize IERSR in a spatially and temporally regulated manner to survive ER-stress or avoid the cytostatic and cytotoxic effects of prolonged IERSR. We further hypothesize that limiting the ability of tumors to activate IERSR or causing sustained and exaggerated IERSR will cause selective demise of tumors. We have developed chemical modulators of the IERSR and genetically engineered human cancer cells lines resistant to these agents. If this proposal is funded, we will utilize our transgenic cell lines and chemical modulators of IERSR to test our hypothesis and to determine conclusively if the IERSR can be pharmacologically targeted for cancer therapy. 1) We will test the hypothesis that N,N'-diarylurea induced sustained eIF2a phosphorylation will inhibit tumor growth. We will study the pharmacokinetic profile and acute toxicity of selected/optimized N,N'-diarylureas. We will determine their efficacy and mechanism specificity by treating mice carrying bilateral tumors expressing eIF2a-WT on one side and non-phosphorylatable mutant, eIF2a-S51A on the other side (Specific Aim 1). 2) We will test the hypothesis that inhibition of Xbp-1 splicing by diaryl-oxindoles inhibit tumor growth by studying the pharmacokinetic profile and acute toxicity of selected/optimized diaryl-oxindole. We will determine their efficacy and mechanism specificity by treating mice carrying bilateral tumors expressing only endogenous Xbp-1 on one side and already spliced Xbp-1 on the other side (Specific Aim 2), and 3) We will test the hypothesis that inhibition of Xbp-1 splicing and induction of eIF2a phosphorylation will synergistically inhibit tumor growth and metastasis (Specific Aim 3). PUBLIC HEALTH RELEVANCE: The purpose of this project is to test the hypothesis that integrated endoplasmic stress response can be pharmaceutically targeted for cancer therapy by treating tumor bearing mice with agents that cause sustained phosphorylation of eIF2a and or inhibition of Xbp-1 splicing individually or in combination. To test this novel hypothesis we have developed small chemical agents as well as breast and prostate cancer cells in which expression of endogenous eIF2a is replaced by recombinant eIF2a (WT or nonphosphorylatable S51A mutant) or cells that express an already spliced Xbp-1 mRNA;these tools enable us to test the efficacy and specificity of small molecular weight agents.